Biodesign for the real world Falco ENZLER and Bastien ORSET General - - PowerPoint PPT Presentation

Biodesign for the real world

Falco ENZLER and Bastien ORSET

General overview

• What is Biodesign?
• What is our kit and why make a new one?
• What about the legal aspects?
• An example of approved field kit: ARSOlux
• What about confinement?
• DNA construct details
• Future Design

2

Biodesign Project

• Biodesign is a multidisciplinary project with different

research aspects :

– development of field compatible water quality sensing systems

– portable real-time sensing – crowd-sourced mapping This project is a collaboration between people from India, Indonesia and Switzerland

• We are students in our third year of life science bachelor at

the EPFL and we joined the Biodesign’s team for our bachelor’s project. http://biodesign.cc/

3

What is our project?

• The aim of this project is to

detect arsenic in water samples in the field.

• This detection is

accomplished with GMO bacteria expressing GFP (Green Fluorescent Protein) in presence of arsenic.

• Our prototype will allow to

quantify GFP i.e. arsenic concentration

4

Why develop a new field kit?

Pros & Cons

5

What is our kit?

• Community based / Understanding of the

prototype

• Reparable

6

What about legal aspects?

• The test kit would use genetically modified

bacteria outside a qualified laboratory

• The main issue is to have the GMO confined

from the environment.

• We thus refer to the « Ordinance on Handling

Organisms in Contained Systems» 814.912

• According to Art.7, our kit is of class 1 i.e. null
• r negligible risk activity.

7

An example of approved GMO field kit: ARSOlux

• Detection of arsenic

via bioluminesence

• Use of a freeze-dried

GMO bioreporter

• Same vial
• Approved by the

German authorities

• Tested in Bangladesh

and other countries

http://www.ufz.de/arsolux/i ndex.php?en=20706

8 http://www.ufz.de/export/data/411/33860_111005_ARSOlux_Projektkennblatt_englisch.pdf Sigfried et al. Environ Sci Technol 2012 46(6) pp.3281-87

What about confinement?

• “contained system means a system that uses physical

barriers or a combination of physical and chemical or biological barriers to limit or prevent contact between

• rganisms and people or the environment “(Art.3,h.)
• After use, Bacteria and antibiotic are destroyed in the lab

by autoclaving or inactivated by hypochlorite solution.

9

Details of the GMO arsenic reporter strain

K-12 E.Coli bacteria strain (similar to ARSOlux:

• E. coli DH5α)
• Can’t do conjugation

due to missing F factor and deficient recA (biological barrier) Plasmid Design

• the plasmid carries

kanamycin resistance, for easy selection

10

Risk Analysis

• What happens if these GMO bacteria are released in

the field? Could Kanamycin resistance and the arsenic reporting system be spread?

• > This E. coli strain cannot transfer the plasmid by itself

to other bacteria. Should cells lyse, the plasmid DNA can be liberated and theoretically be taken up by naturally transformable strains.

• GFP non toxic and its fluorescence is not dangerous for

the cell.

• Other drugs exist to kill Kanamycin resistant bacteria.
• > This resistance could be removed but the detection

efficiency would decrease.

11

Antibiogram

12

Future Design & Possibilities

• Turbidity measurement to normalize the GFP

signal

• Electronic system incorporated inside the box
• Built-in interface (buttons, screen…)
• Use with a smartphone via an application
• Adaptable to other bioreporters for harmful

elements detection (Mercury etc…)

• Field test

13

14

Answer to possible questions

15

Why As is not good? Where?

• Interfere with Krebs cycle (inhibits pyruvate

conversion to acetyl-coA)

• As a slow poison, causes diseases

– Skin diseases – Intestinal tract problems – Cancers

• Maximum concentration advised by WHO:

10μg/L

– Letal dose: 1mg/kg/day

• Problem in Bangladesh and some Asian countries

What is our current cost?

• Around 50 CHF
• Possibility to develop a cheaper prototype

using another micro-controller, i.e a simpler Arduino hardware.

17

Why not detect bioluminescence?

18

Difference between arsenite and arsenate?

• Arsenite has less oxygen than Arsenate

19

Arsenite AsIII (As2O3, AsO2

• , AsO3

3-)

Reporter more sensitive Arsenate AsV (AsO4

3-)